Effects of acute and chronic disease on cell junctions in mouse liver

Cell junctions, including anchoring, occluding and communicating junctions, play an indispensable role in tissue architecture and homeostasis. Consequently, malfunctioning of cell junctions is linked with a wide range of disorders, including in liver. The present study was set up to investigate the effects of acute and chronic disease induced by chemical compounds on hepatic cell junctions in mice. Mice were either overdosed with paracetamol or repeatedly administered carbon tetrachloride followed by sampling at 24 hours or 8 weeks, respectively. mRNA and protein expression levels of adherens, gap and tight junction components were measured in liver using reverse transcription quantitative real-time polymerase chain reaction analysis and immunoblot techniques, respectively. It was found that protein levels of the adherens junction building blocks β-catenin and γ-catenin, the gap junction components Cx26 and Cx32, and the tight junction constituent zonula occludens 2 were decreased, while mRNA levels of the adherens junction building block E-cadherin, and the tight junction constituent zonula occludens 2 and claudin 1 were upregulated following paracetamol overdosing. Repeated administration of carbon tetrachloride increased protein levels of E-cadherin, β-catenin, Cx26, Cx32, Cx43 and claudin 1. The latter was reflected at the mRNA level. In conclusion, acute and chronic liver disease have different effects on cell junctions in liver.


INTRODUCTION
Cell junctions play an important role in maintaining tissue architecture and homeostasis (Garcia et al., 2017;Adil et al., 2021). These multiprotein complexes are embedded in the cell plasma membrane and can be classified according to their function, including anchoring, occluding and communicating junctions (Samiei et al., 2019). Anchoring or adherens junctions establish adhesion between neighboring cells. They provide a mechanistic linkage and are built up by cytoplasmic cadherins and cytosolic catenins (Troyanovsky et al., 2021). In this respect, the E-cadherin protein is seen as a guardian of liver physiology. E-cadherin, which is expressed by hepatocytes and biliary epithelial cells, is an important regulator of bile flow in the intrahepatic biliary network (Gonzalez-Sanchez et al., 2015). β-catenin and γ-catenin are also produced by hepatocytes and maintain hepatocyte structure and function (Wickline et al., 2011;Pradhan-Sundd et al., 2018). β-catenin also participates in the canonical Wnt pathway, which controls the expression of genes involved in glutamine synthesis and biotransformation (Sekine et al., 2006). Occluding junctions or tight junctions regulate cell polarity and form a permeability barrier to control the passage of ions and solutes into the paracellular space (Tsukita et al., 2019;Krug and Fromm 2020). Like adherens junctions, tight junctions are composed of transmembrane proteins and cytoplasmic proteins. Transmembrane components, including claudins, occludin, junctional adhesion molecules and the coxsackie-adenovirus receptor, provide the interaction between adjacent cells. These membrane-spanning proteins are tethered to cytoskeletal-proteins, such as zonula occludens, cingulin and symplekin proteins, to connect the cytoskeleton of neighboring cells (Shi et al., 2018;Tsukita et al., 2019;Lynn et al., 2020). Functional tight junctions are required for normal liver functionality, in particular bile flow. Zonula occludens 2 and claudin 1 are indeed critical for the establishment of the blood-bile barrier and the control of paracellular permeability, respectively (Hadj-Rabia et al., 2004;Grosse et al., 2012;Itoh et al., 2021;Xu et al., 2021). Communicating or gap junctions are composed of 2 connexin (Cx) hemichannels of 2 adjacent cells, which in turn are built up by 6 Cx proteins. Gap junctions form waterfilled tunnels that allow the diffusion of small and hydrophilic molecules between the cytosol of adjacent cells (Van Campenhout et al., 2022). Cx26 and Cx32 are mainly expressed by hepatocytes, while most of the non-parenchymal liver cells harbor Cx43. Gap junctions, in particular Cx32-based channels, support liver-specific functions, including biotransformation, bile production, synthesis and secretion of proteins and control of carbohydrate metabolism (Van Campenhout et al., 2022). Since adherens, tight and gap junctions are goalkeepers of hepatic homeostasis, it is not surprising that these structures are frequently implicated in liver disease (Garcia et al., 2017;Adil et al., 2021). The present study was set up in order to investigate the effects of acute and chronic liver disease on adherens, tight and gap junctions in liver. To mimic acute liver disease, mice were overdosed with paracetamol (PAR) for 24 hours, while chronic liver disease was simulated by repeated administration of carbon tetrachloride (CCL4) to mice for 8 weeks.

Animals and treatment
Male C57BL/6 mice (Jackson Laboratories, USA) were housed in the animal facility of the School of Veterinary Medicine and Animal Science of the University of São Paulo, Brazil. Animals were kept under controlled environmental conditions, in particular in a room with ventilation (16-18 air changes/ hour), relative humidity (45-65 %), controlled temperature (20-24 °C) and light/dark cycle 12:12, and were given water and balanced diet (Nuvital Nutrientes LTDA, Brazil) ad libitum. In the first set of experiments, mice were starved 15-16 hours prior to administration of PAR or 0.9 % sodium chloride (SAL) solution. PAR was dissolved in saline solution, slightly heated and injected (30-37 °C) intraperitoneally at 300 mg/kg body weight. After injection, mice regained free access to food. Mice were euthanized 24 hours following PAR overdosing by exsanguination during sampling under isoflurane-induced anesthesia. Another group of male C57BL/6 mice were administered corn oil (OIL) or CCL4 at a gradually increased dose for 8 weeks. Animals received 3 intraperitoneally injections weekly. The initial dose of CCL4 was 0.25 mg/kg body weight, and there were 0.25 mg weekly increments to the utmost dose of 1.25 mg/kg body weight. Mice were euthanized after completion of OIL or CCL4 treatment by exsanguination during sampling under isoflurane-induced anesthesia. Mouse blood, collected by cardiac puncture, was drawn into a heparinized syringe and centrifuged for 10 minutes at 1503×g, and serum was stored at -20 °C. Livers were snap-frozen in liquid nitrogen with storage at -80 °C. Both studies have been approved by the Committee on Bioethics of the School of Veterinary Medicine and Animal Science of the University of São Paulo, Brazil and all animals received humane care according to the criteria outlined in the "Guide for the Care and Use of Laboratory Animals".

Immunoblot analysis
Immunoblot analysis of liver tissue was performed as previously described with slight modifications . In short, liver tissue was homogenized in radioimmunoprecipitation assay buffer containing Halt Protease Inhibitor Cocktail (Thermo Scientific, USA). Homogenates were centrifuged at 14000×g for 20 minutes at 4 °C. Protein concentrations in supernatants were determined using a commercial Pierce BCA Protein Assay kit (Thermo Scientific, USA). Protein lysates of 25 µg were resolved on sodium dodecyl sulphate polyacrylamide stain-free gels (Bio-Rad, USA) under reducing conditions. Separated proteins were transferred to nitrocellulose membranes (Bio-Rad, USA) and blocking was performed with 5 % milk in Tris-buffered saline solution containing 0.1 % Tween 20. Membranes were incubated overnight at 4 °C with primary antibody directed against E-cadherin, β-catenin, γcatenin, Cx26, Cx32, Cx43, zonula occludens 2 and claudin 1 (Supplementary Table 1) followed by incubation for 1 hour at room temperature with a secondary HRP-conjugated goat anti-rabbit IgG antibody (1:1000) (Dako, USA). Detection of the proteins was carried out by means of enhanced chemiluminescence. For semi-quantification purposes, a normalization method based on total protein loading was used. Stain-free technology introduces the binding of a trihalo compound to tryptophan residues present in the sample. This allows visualization of total protein signals with low background and overcomes the drawbacks of housekeeping proteins (Diller et al., 2021). All cell junction proteins were normalized against total protein loading and expressed as relative alterations compared to SAL-treated and OIL-treated animals for the acute and chronic liver disease model, respectively.

Reverse transcription quantitative real-time polymerase chain reaction analysis
Reverse transcription quantitative realtime polymerase chain reaction (RT-qPCR) analysis of liver tissue was performed as explained elsewhere (Maes et al., 2016c). Briefly, total RNA was isolated from liver tissue using a GenElute Mammalian Total RNA Purification Miniprep Kit (Sigma, USA) and the On-Column DNase Digestion Set (Sigma, USA) according to the manufacturer's instructions. A NanoDrop ND-100 Spectrophotometer (Thermo Scientific, USA) was used to control purity and quantify RNA. Next, 2 µg RNA was reversely transcribed into cDNA with an iScript cDNA Synthesis Kit (Bio-Rad, USA) using an iCycler iQ (Bio-Rad, USA) followed by cDNA purification using a GenElute PCR Clean-Up Kit (Sigma, USA). cDNA products were quantitatively amplified using Taqman probes and primers specific for target and reference genes (Thermo Scientific, USA) (Supplementary Table 2). After running the samples in duplicate in a Quant-Studio PCR System (Thermo Scientific, USA), the system's software was used to calculate quantification cycles. Stable reference genes for normalization were determined by geNorm using the qbase + software (Biogazelle, Belgium) (Vandesompele et al., 2002). The resulting ΔCq values of the test samples were normalized to those of the cali-brator samples, yielding ΔΔCq values. Relative alterations (fold change) in RNA levels were calculated according to the 2 (-ΔΔCq) formula (Livak and Schmittgen, 2001). Results were expressed as fold change of ΔΔCq values obtained from SAL-treated and OILtreated mice.

Statistical analysis
All data were expressed as mean ± standard deviation (SD). The number of biological replicates (n) and technical replicates (N) are specified for each analysis in the figure legends. Results were statistically processed by 2-tailed unpaired student t-tests and Welch's correction using GraphPad Prism7 software, with probability (p) values of less than or equal to 0.05 considered as significant.

Figure 2:
Effect of acute liver disease on mRNA levels of hepatic cell junction components. Mice were injected with SAL or PAR solution. After 24 hours, mRNA was extracted from the liver and subjected to RT-qPCR analysis of E-cadherin, β-catenin, γ-catenin, Cx26, Cx32, Cx43, zonula occludens 2 and claudin 1. Fold changes in RNA levels were calculated, where the average expression of SALtreated animals was set to 1. Results were analyzed by 2-tailed unpaired student t-tests and Welch's correction. Data were expressed as means ± SD (*p ≤ 0.05) (n = 4-5; N = 2).

Figure 4: Effect of chronic liver disease on mRNA levels of hepatic cell junction components.
Mice received injections with OIL or CCL4 for 8 weeks. mRNA was extracted from the liver and subjected to RT-qPCR analysis of E-cadherin, β-catenin, γ-catenin, Cx26, Cx32, Cx43, zonula occludens 2 and claudin 1. Fold changes in mRNA levels were calculated, where the average expression of OIL-treated animals was set to 1. Results were analyzed by 2-tailed unpaired student t-tests and Welch's correction. Data were expressed as means ± SD (*p ≤ 0.05) (n = 8-10; N = 2).

DISCUSSION
The present study was set up in order to investigate the effects of acute and chronic liver experimentally induced in mice by exposure to chemical compounds on the expression of hepatic cell junction components. Given that anchoring, occluding and communicating junctions support liver homeostasis, hepatic cell junction components are often targeted in liver disease. The acute liver disease model used in this study relied on PAR overdosing and caused a reduced expression of β-catenin, γ-catenin, Cx26, Cx32 and zonula occludens 2 proteins in mouse liver. In vitro and in vivo studies previously demonstrated the malfunctioning of liver cell junctions upon PAR intoxication (Newsome et al., 2004;Maes et al., 2016a;Gamal et al., 2017). Likewise, PAR evoked disruption of tight junctions accompanied by a dose-dependent loss of zonula occludens 1 in cultures of human hepatoma cells and primary mouse hepatocytes (Gamal et al., 2017). Furthermore, human hepatoma cells and primary human hepatocytes lost their adhesive capacity when exposed to serum of patients with PAR induced acute liver failure (Newsome et al., 2004). Overdosing mice with PAR also deteriorated gap junction activity and decreased Cx32 protein levels (Maes et al., 2016a). Given that mRNA levels of E-cadherin, zonula occludens 2 and claudin 1 were upregulated in the present study, the effects of PAR on hepatic cell junction proteins seemed not be mediated by the transcriptional machinery.
Malfunctioning of cell junctions and loss of hepatic cell junction components is associated with several other liver diseases. In this respect, downregulation of the expression of tight junction components is frequently linked with chronic biliary disease (Roehlen et al., 2020). Similarly, the production of zonula occludens 1, occludin and claudin 1, is negatively affected in liver fibrosis (Wang et al., 2021). mRNA and protein expression levels of most of these cell junction components are also decreased in cholestasis (Huang et al., 2021). Furthermore, liver injury leads to dysfunction of gap junctions accompanied with changes in Cx26, Cx32 and Cx43 quantities (Yang et al., 2019;Van Campenhout et al., 2022). Accordingly, hepatic Cx32 expression is low in patients with non-alcoholic steatohepatitis and negatively correlates with increasing liver injury (Luther et al., 2018). On the other hand, hepatitis C virus infection and hepatocellular carcinoma can cause the upregulation of hepatic tight junction protein expression (Roehlen et al., 2020). Bile duct ligation-induced liver injury drastically increased adherens junction component quantities in mouse liver, which might be caused by hepatic repair processes (Li et al., 2007;Clerbaux et al., 2019;Van Campenhout et al., 2019). Repeated administration of CCL4 was used to initiate chronic liver disease in mice in the present study. Hepatic protein levels of E-cadherin, β-catenin, Cx26, Cx32, Cx43 and claudin 1 were elevated in CCL4-treated mice. This might be related to type 2 epithelial to mesenchymal transition (EMT). Despite some controversy, type 2 EMT appears to be involved in liver fibrosis (Choi and Diehl, 2009;Xie and Diehl, 2013;Zhao et al., 2016). During EMT, epithelial cells acquire a migratory character, start to express mesenchymal markers, like β-catenin, and evolve into collagen producing fibroblasts (Zhao et al., 2016). Cells undergoing EMT do not only produce mesenchymal markers, but they also secure their epithelial nature by expressing epithelial-specific E-cadherin (Kalluri and Weinberg, 2011). In this regard, advanced type 2 EMT activity might underly the increased expression of E-cadherin and βcatenin. In accordance with the surge in claudin 1 at the transcriptional and translational level, CCL4 has been reported to manifest an increase in hepatic tight junction components contributing to hepatic sinusoidal resistance and portal hypertension (Hintermann et al., 2016). While there was no difference in Cx26, Cx32 and Cx43 mRNA expression in fibrotic mice, chronic liver disease induced expression of the corresponding proteins. Genetic ablation of Cx32 proteins revealed a role for Cx32 in tissue protection during liver fibrosis. Enhancement of Cx32 production may counteract hepatocyte damage and protect against chronic liver injury . In addition, it has been reported that induced expression of hepatic Cx43 proteins in fibrotic mice has a function in the control and regulation of fibrogenesis. Cx43-deficient mice developed excessive liver fibrosis after repeated administration of CCL4 (Cogliati et al., 2011). As a general conclusion, the present study showed that acute and chronic liver disease have different effects on cell junctions in liver.